One of the most critical aspects in optical microscopy is to ensure the specimen is illuminated with light that is bright, glare-free, and evenly dispersed in the field of view. Discussions about microscope illumination cover the wide spectrum of light sources available for optical microscopy, the theory of Köhler illumination, and the practical aspects of adjusting a microscope for proper illumination in both transmitted and reflected light.
Introduction to Microscope Illumination - All to frequently, sophisticated and well-equipped microscopes fail to yield excellent images due to incorrect use of the light source, which usually leads to inadequate sample illumination. When optimized, illumination of the specimen should be bright, glare-free, and evenly dispersed in the field of view. Presented in this section is an overview of microscope illumination, including a discussion of the Critical illumination technique, which was often employed prior to the widespread acceptance of Köhler illumination.
Light Sources for Optical Microscopy - Modern microscopes usually have an integral light source that can be controlled to a relatively high degree. The most common source for today's microscopes is an incandescent tungsten-halogen bulb positioned in a reflective housing that projects light through the collector lens and into the substage condenser. Other sources include arc-discharge lamps, light emitting diodes (LEDs), and lasers.
Köhler Microscope Illumination - Illumination of the specimen is the most important variable in achieving high-quality images in microscopy and critical photomicrography. The Köhler illumination technique was first introduced in 1893 by August Köhler of the Carl Zeiss corporation as a method of providing the optimum specimen illumination. This technique is recommended by all manufacturers of modern laboratory microscopes because it can produce specimen illumination that is uniformly bright and free from glare, thus allowing the user to realize the microscope's full potential.
Illumination with Transmitted Light - Adjustment of a transmitted light microscope for Köhler illumination is a logical and relatively easy process that should be practiced until mastered by all serious students of microscopy. Each time a microscope is turned on, it should be carefully inspected to ensure proper alignment of all optical components, and to guarantee that the lamp is centered and the condenser and field diaphragm are properly adjusted for Köhler illumination.
Illumination with Reflected Light - In brightfield reflected light microscopy (including contrast enhancing techniques such as differential interference contrast and darkfield), proper use of the two variable diaphragms enable the use of the highly desirable Köhler illumination. It is important to note, that in reflected light systems, the objective serves a dual function: on the way to the specimen as a matching well-corrected condenser properly aligned; on the way back to the microscope as an image-forming objective in the customary role of an objective projecting the image-carrying rays toward the eyepiece.
August Köhler's Original Manuscript - Translated from German, the original article appearing in Zeitschrift für wissenschaftl. Mikroskopie, was reprinted in the Köhler Illumination Centenary commemorative issue by the Royal Microscopical Society in 1994. Included in the article is the original woodcut drawing that depicts the microscope optical train, as well as a re-labeled color version for added clarity. Dr. Köhler entitled the article "A New System of Illumination for Photomicrographic Purposes" because of the importance that his unique illumination technique afforded to recording images on film emulsions in the early twentieth century. Köhler illumination is still the method of choice for microscope illumination in traditional film photomicrography and newer digital imaging techniques.
Fluorescence Microscopy Light Sources - In order to generate enough excitation light intensity to furnish secondary fluorescence emission capable of detection, powerful light sources are needed. These are usually either mercury or xenon arc (burner) lamps, which produce high-intensity illumination powerful enough to image faintly visible fluorescence specimens.
Laser Systems for Optical Microscopy - The lasers commonly employed in optical microscopy are high-intensity monochromatic light sources, which are useful as tools for a variety of techniques including optical trapping, lifetime imaging studies, photobleaching recovery, and total internal reflection fluorescence. In addition, lasers are also the most common light source for scanning confocal fluorescence microscopy, and have been utilized, although less frequently, in conventional widefield fluorescence investigations.
Focusing and Alignment of Arc Lamps - Mercury and xenon arc lamps are now widely utilized as illumination sources for a large number of investigations in widefield fluorescence microscopy. Visitors can gain practice aligning and focusing the arc lamp in a Mercury or Xenon Burner with this Nikon MicroscopyU interactive tutorial, which simulates how the lamp is adjusted in a fluorescence microscope.
Light Emitting Diode Fundamentals - The past few decades have brought a continuing and rapidly evolving sequence of technological revolutions, particularly in the digital arena, which has dramatically changed many aspects of our daily lives. The developing race among manufacturers of light emitting diodes (LEDs) promises to produce, literally, the most visible and far-reaching transition to date. Recent advances in the design and manufacture of these miniature semiconductor devices may result in the obsolescence of the common light bulb, perhaps the most ubiquitous device utilized by modern society.
Interactive Java Tutorials
These tutorials explore various aspects in preparing a microscope for Köhler illumination, and allow students to practice alignment of the microscope without the burden of requiring the presence of a physical instrument.
Microscope Alignment for Köhler Illumination - Perhaps one of the most misunderstood and often neglected concepts in optical microscopy is proper configuration of the microscope with regards to illumination, which is a critical parameter that must be fulfilled in order to achieve optimum performance. The intensity and wavelength spectrum of light emitted by the illumination source is of significant importance, but even more essential is that light emitted from various locations on the lamp filament be collected and focused at the plane of the condenser aperture diaphragm. This MicroscopyU interactive tutorial reviews both the filament and condenser alignment procedures necessary to achieve Köhler illumination.
Condenser Alignment - Learn to align the most important optical components in a microscope with this tutorial. Students can practice centering the condenser and setting the field diaphragm to its optimum size for photomicrography.
Condenser Light Cones - This tutorial explores how changes in the size of the condenser aperture affect the shape and size of the light cone (and numerical aperture) emitted from the condenser.
Condenser Aperture Diaphragm Adjustment - Discover how the aperture diaphragm of the substage condenser can be monitored with a Bertrand lens (or by removing the eyepiece) to optimize numerical aperture, image resolution, depth of field, and overall image quality.
Conjugate Planes - Discover how various conjugate plane sets pass through focus together when a microscope is properly aligned for optimum Köhler illumination.
Condenser Aperture Diaphragm Control Of Specimen Contrast - This tutorial explores how condenser aperture diaphragm opening size affects specimen contrast at medium to high (10x through 100x) magnifications.
Lamp Filament Alignment - Alignment of the lamp filament is one of the first and most crucial steps in achieving proper Köhler illumination. This tutorial explores all of the aspects of adjusting both the voltage and the position of the lamp filament, and ensuring that the filament completely fills the back focal plane of the objective.
Substage Condenser Numerical Aperture - The condenser aperture diaphragm controls the numerical aperture of the optical system of a microscope. This tutorial demonstrates changes in condenser light cones as a function of numerical aperture settings.
Eyepiece Diopter Adjustment - Eyepieces in modern microscopes have both interpupillary and individual diopter adjustments that help adjust the eyepieces for comfort and to correct for near and far sightedness.
Contrast Adjustment At Low Magnifications - Specialized condensers suited for low magnification (5x objectives and less) microscopy rely on the field diaphragm for adjustment of specimen contrast. Some general purpose condensers have a swing-out lens for use with low magnification objectives, and these also rely on the field diaphragm.
Aperture and Field Diaphragm Adjustment in Reflected Light - Reflected light microscopes are adjusted for Köhler illumination in a manner similar to transmitted light microscopes. This tutorial examines how the aperture iris diaphragm is used to vary specimen contrast and image quality, and how the field diaphragm is manipulated for optimum reduction of glare and scattered light.
Reflected Light Illuminators - Discover how light travels through a reflected light illuminator with this interactive Java tutorial.
Significant contributions to the theory and practice of microscope illumination were made by the pioneers in optics listed in the links below. Among these scientists are Ernst Abbe, inventor of the achromatic objective, August Köhler, who developed an illumination technique that is still widely employed, and Johannes Lieberkuhn, inventor of a unique reflector for incident light illumination.
Ernst Abbe (1840-1905) - Ernst Abbe was a brilliant German mathematician and physicist who made several of the most important contributions to the design of lenses for optical microscopy. Abbe studied physics and mathematics as an undergraduate at the University of Jena and went to graduate school at the University of Göttingen, where he received a doctorate in thermodynamics. In 1863 Abbe joined the faculty at the University of Jena where he taught physics. He met Carl Zeiss in 1866 and became very interested in the optical problems surrounding mid-nineteenth century microscopy. Together with Zeiss, Abbe formed a partnership and he was made the research director of Zeiss Optical Works late in 1866, and assumed control of the company when Zeiss died in 1888.
August Köhler (1866-1948) - August Köhler, a German scientist and photomicrographer born in 1866, is best known for his development of a superior microscope illumination technique, which is still utilized today, and for designing the first ultraviolet microscope. The method, called Köhler illumination, is also known as double diaphragm illumination, because it uses both a field and an aperture iris diaphragm to configure microscope illumination. Setting up the light path correctly with this configuration results in an evenly illuminated field of view and a brighter image without glare.
Johannes Nathaniel Lieberkuhn (1711-1756) - Johannes Nathaniel Lieberkuhn was a German physician, anatomist, and physicist. He is most widely known for development of the solar microscope, studies of the intestine, and invention of a reflector for improving microscopic viewing of opaque specimens. He was also a member of the mathematics department at the Berlin Academy of Sciences and created a lens that enhanced the use of early portable microscopes for botanical fieldwork.
John Thomas Quekett (1815-1861) - Inspired by Joseph Jackson Lister's 1830 paper on achromatic microscopes, Quekett and his brother Edwin were among the seventeen founding members of the Microscopical Society of London, now known as the Royal Microscopical Society. As the world's first microscopical organization, formation of the group was of great consequence and has resulted in a significant impact on many fields related to microscopy. They began humbly, however, in 1839 at Edwin's house, Number 50 Wellclose Square in London.
Selected Literature References
Selected References on Microscope Illumination - Gathered from our vast library of literature on optical microscopy, the reference materials listed in this section are an excellent source of additional information on microscope illumination. Included in this section are references to review articles and original research reports that discuss various aspects of the theory and applications regarding how microscopes are configured to take advantage of various illumination scenarios. In addition, basic articles describing the properties of a variety of natural and artificial light sources are included.
Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.
Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.
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